Despite the widely observed predominance of Cand . Patescibacteria in subsurface communities, their input source and ecophysiology are poorly understood. Here we study mechanisms of the formation of a groundwater microbiome and the subsequent differentiation of Cand . Patescibacteria. In the Hainich Critical Zone Exploratory, Germany, we trace the input of microorganisms from forested soils of preferential recharge areas through fractured aquifers along a 5.4 km hillslope well transect. Cand . Patescibacteria were preferentially mobilized from soils and constituted 66% of species-level OTUs shared between seepage and shallow groundwater. These OTUs, mostly related to Cand . Kaiserbacteraceae, Cand . Nomurabacteraceae, and unclassified UBA9983 at the family level, represented a relative abundance of 71.4% of the Cand . Patescibacteria community at the shallowest groundwater well, and still 44.4% at the end of the transect. Several Cand . Patescibacteria subclass-level groups exhibited preferences for different conditions in the two aquifer assemblages investigated: Cand . Kaiserbacteraceae surprisingly showed positive correlations with oxygen concentrations, while Cand . Nomurabacteraceae were negatively correlated. Co-occurrence network analysis revealed a central role of Cand . Patescibacteria in the groundwater microbial communities and pointed to potential associations with specific organisms, including abundant autotrophic taxa involved in nitrogen, sulfur and iron cycling. Strong associations among Cand . Patescibacteria themselves further suggested that for many groups within this phylum, distribution was mainly driven by conditions commonly supporting a fermentative life style without direct dependence on specific hosts. We propose that import from soil, and community differentiation driven by hydrochemical conditions, including the availability of organic resources and potential hosts, determine the success of Cand . Patescibacteria in groundwater environments.

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either 13 C-labeled benzene or 13 C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of 13 C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities. The ISME Journal (2012) 6, 2291-2301; doi:10.1038/ismej.2012.68; published online 12 July 2012 Subject Category: microbial ecology and functional diversity of natural habitats

We propose a joint experimental and theoretical approach to the automated reconstruction of elemental fluxes in microbial communities. While stable isotope probing of proteins (protein-SIP) has been successfully applied to study interactions and elemental carbon and nitrogen fluxes, the volume and complexity of mass spectrometric data in protein-SIP experiments pose new challenges for data analysis. Together with a flexible experimental setup, the novel bioinformatics tool MetaProSIP offers an automated high-throughput solution for a wide range of (13)C or (15)N protein-SIP experiments with special emphasis on the analysis of metaproteomic experiments where differential labeling of organisms can occur. The information calculated in MetaProSIP includes the determination of multiple relative isotopic abundances, the labeling ratio between old and new synthesized proteins, and the shape of the isotopic distribution. These parameters define the metabolic capacities and dynamics within the investigated microbial culture. MetaProSIP features a high degree of reproducibility, reliability, and quality control reporting. The ability to embed into the OpenMS framework allows for flexible construction of custom-tailored workflows. Software and documentation are available under an open-source license at www.openms.de/MetaProSIP.

). (2015) XoxFencoding an alternative methanol dehydrogenase is widespread in coastal marine environments. Environmental Microbiology, 17 (10). pp. 3937-3948. Permanent WRAP URL:http://wrap.warwick.ac.uk/67990 Copyright and reuse:The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.Copies of full items can be used for personal research or study, educational, or not-for profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Publisher's statement:"This is the peer reviewed version of the following article: which has been published in final form at http://dx.doi.org/10.1111/1462-2920.12896 This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." A note on versions:The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher's version. Please see the 'permanent WRAP url' above for details on accessing the published version and note that access may require a subscription. Abstract 22The xoxF gene, encoding a pyrroloquinoline quinone-dependent methanol dehydrogenase, is found in all 23 known proteobacterial methylotrophs. In several newly discovered methylotrophs, XoxF is the active methanol 24 dehydrogenase, catalysing the oxidation of methanol to formaldehyde. Apart from that, its potential role in 25 methylotrophy and carbon cycling is unknown. So far, the diversity of xoxF in the environment has received 26 little attention. We designed PCR primer sets targeting clades of the xoxF gene, and used 454 pyrosequencing 27 of PCR amplicons obtained from DNA of four coastal marine environments for a unique assessment of the 28 diversity of xoxF in these habitats. Phylogenetic analysis of the data obtained revealed a high diversity of xoxF 29 genes from two of the investigated clades, and substantial differences in sequence composition between 30 environments. Sequences were classified as being related to a wide range of both methylotrophs and non-31 methylotrophs from Alpha-, Beta-and Gammaproteobacteria. The most prominent sequences detected were 32 related to the family Rhodobacteraceae, the genus Methylotenera and the OM43 clade of Methylophilales, 33 and are thus related to organisms that employ XoxF for methanol oxidation. Furthermore, our analyses 34 revealed a high degree of so far undescribed sequences, suggesting a high number of unknown species in 35 these habitats. 36